Human immunodeficiency virus type 1 (HIV-1) encodes the transactivating protein Tat, which is essential for virus replication and progression of HIV disease. Tat has multiple domains and consequently the molecular mechanisms by which Tat regulates viral and cellular gene expression are complex. The HIV epidemic remains one of the greatest threats to public health, in part because effective vaccines or antiviral agents have been difficult to develop. Although viral reverse transcriptase and protease inhibitors reduce the level of virus replication, the benefit is, in most cases, short term because of the emergence of resistant viral strains. Tat transactivation is an attractive target for antiviral therapy because Tat is required for viral gene expression not only during exponential growth, but also during activation of the integrated proviral genomes that give rise to drug-resistant strains. Tat stimulates human immunodeficiency virus type 1 (HIV-1) transcriptional elongation by recruitment of cellular transcription elongation factor, P-TEFb, to the HIV-1 elongation complex. Tat transactivation is also critically dependent upon the recruitment of cellular transcription factors SPT-5 and Tat-SF1. Using an immobilized DNA template assay, we have analyzed the effect of Tat on P-TEFb kinase activity during the initiation and elongation phases of HIV-1 transcription. Results demonstrate that Tat/TAR-dependent CDK9 kinase activity regulates phosphorylation of RNAP II CTD Ser 5 as well as the transcription factors SPT5 and Tat-SF1 to stimulate transcription elongation of HIV-1 promoter. Utilizing chromatin immunoprecipitation assays of HIV-1-infected cells, we show that a low concentration of flavopiridol inhibits Ser 5 phosphorylation of RNAP II CTD and association of SPT 5 with transcriptionally induced HIV-1 gene in vivo. RNAi knockdown of SPT5 and Tat-SF1 attenuated HIV-1 replication, showing dependence of viral replication on the transcription elongation factors SPT5 and Tat-SF1. These observations argue that Tat modifies CDK9 kinase activity which is crucial for HIV-1 transcription elongation and that selective inhibition of the Tat cofactors could be an important target to block viral replication. Tat-induced Ser 5 phosphorylation of CTD by P-TEFb also stimulates the guanylyltransferase activity of the human capping enzyme (HCE) and RNA cap formation. Sequential phosphorylation of CTD by Tat-induced P-TEFb enhances the stimulation of HCE guanylyltransferase activity and RNA cap formation by TFIIH-mediated CTD phosphorylation. Using an immobilized template assay that permits isolation of transcription complexes, we show that Tat/TAR-dependent phosphorylation of RNAP II CTD stimulates cotranscriptional capping of HIV-1 mRNA. Upon transcriptional induction of latently infected cells, capped transcripts along with Ser 5-phosphorylated RNAP II accumulate in the promoter proximal region of the HIV-1 genome. These observations suggest that Tat/TAR-dependent phosphorylation of RNAP II CTD is crucial not only in promoting transcription elongation but also in stimulating nascent viral RNA capping.